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Fibrin(Ogen) As a Therapeutic Target: Opportunities and Challenges

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Fibrin(Ogen) As a Therapeutic Target: Opportunities and Challenges International Journal of Molecular Sciences Review Fibrin(ogen) as a Therapeutic Target: Opportunities and Challenges Thembaninkosi G. Gaule and Ramzi A. Ajjan * Division of Cardiovascular & Diabetes Research, Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), University of Leeds, Leeds LS2 9JT, UK; [email protected] * Correspondence: [email protected] Abstract: Fibrinogen is one of the key molecular players in haemostasis. Thrombin-mediated release of fibrinopeptides from fibrinogen converts this soluble protein into a network of fibrin fibres that form a building block for blood clots. Thrombin-activated factor XIII further crosslinks the fibrin fibres and incorporates antifibrinolytic proteins into the network, thus stabilising the clot. The conversion of fibrinogen to fibrin also exposes binding sites for fibrinolytic proteins to limit clot formation and avoid unwanted extension of the fibrin fibres. Altered clot structure and/or incorporation of antifibrinolytic proteins into fibrin networks disturbs the delicate equilibrium between clot formation and lysis, resulting in either unstable clots (predisposing to bleeding events) or persistent clots that are resistant to lysis (increasing risk of thrombosis). In this review, we discuss the factors responsible for alterations in fibrin(ogen) that can modulate clot stability, in turn predisposing to abnormal haemostasis. We also explore the mechanistic pathways that may allow the use of fibrinogen as a potential therapeutic target to treat vascular thrombosis or bleeding disorders. Better understanding of fibrinogen function will help to devise future effective and safe therapies to modulate thrombosis and bleeding risk, while maintaining the fine balance between clot formation and lysis. Keywords: fibrinogen; fibrin; fibrinolysis; thrombosis; hyperfibrinolysis; therapeutics Citation: Gaule, T.G.; Ajjan, R.A. Fibrin(ogen) as a Therapeutic Target: Opportunities and Challenges. Int. J. Mol. Sci. 2021, 22, 6916. https:// 1. Introduction doi.org/10.3390/ijms22136916 Fibrinogen is one of the most abundant plasma proteins, circulating at 2–3 mg/mL Academic Editor: Nobuo Okumura concentrations, but levels can more than double in pathological states [1,2]. Soluble fibrinogen is converted into an insoluble fibrin network, which forms the backbone of the Received: 5 June 2021 blood clot and has a critical role in haemostasis by limiting blood loss following vascular Accepted: 24 June 2021 injury [3]. However, in diseased blood vessels, the rupture of an atheromatous plaque can Published: 28 June 2021 trigger pathological clot formation, which, in severe cases, blocks the vessel, causing end organ damage including myocardial infarction and stroke. Publisher’s Note: MDPI stays neutral Quantitative and qualitative changes in fibrinogen can result in fibrin networks that with regard to jurisdictional claims in are difficult to breakdown [4], thus increasing the risk of thrombosis and vascular occlusion. published maps and institutional affil- Other alterations in fibrinogen can result in ineffective or unstable fibrin networks, thus in- iations. creasing the risk of bleeding [5]. Therefore, the manipulation of the fibrinogen molecule has the potential to alter thrombosis or bleeding risk by inhibiting clot formation/facilitating lysis or by making clots that are resistant to breakdown. While the fibrin network is targeted to treat vascular occlusion, there is no treatment Copyright: © 2021 by the authors. directed at the fibrinogen molecule itself. The same applies for conditions associated with Licensee MDPI, Basel, Switzerland. blood loss; fibrin sealants, composed of a mixture of coagulation proteins, have been used This article is an open access article to reduce bleeding following vascular injury [6], but again, the fibrinogen molecule is not distributed under the terms and used as a target for bleeding disorders. conditions of the Creative Commons In the current review, we describe the process of clot formation and lysis, discuss the Attribution (CC BY) license (https:// factors responsible for stabilising the fibrin network and explore the potential role of the creativecommons.org/licenses/by/ fibrinogen molecule as a therapeutic target. 4.0/). Int. J. Mol. Sci. 2021, 22, 6916. https://doi.org/10.3390/ijms22136916 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 6916 2 of 20 Structure of Fibrinogen Fibrinogen circulates in blood as a 340 KDa soluble homodimeric glycoprotein. Each subunit comprises of three polypeptide chains: Aα,Bβ, and γ encoded by three genes, FGA, FGB and FGG, respectively, which are located in a 3-gene cluster on human chromosome 4 [7]. The Aα and Bβ chains are constitutively expressed with their expression regulated by housekeeping mechanisms so as to maintain the levels of circulating fibrinogen in the blood [8,9]. The Bβ chain is transcribed from eight exons and encodes for one form of the Bβ chain. The Aα is transcribed from five exons, however, alternative splicing from a sixth exon encodes for AαE chain, which accounts for 1–3% of circulating fibrinogen [7]. Similar to the Aα chain, γ chains exist in two forms: γ and γ0. The major γ chain is transcribed from ten exons, while intron 9 is retained in γ0 making its C-terminus 20 amino acids longer than the γ chain. Fibrinogen molecules containing γ’ exist as heterodimers γ/γ’ or homodimers γ0/γ0 accounting for 8–15% and <1%, respectively, of the total circulating fibrinogen in healthy individuals [7,10]. The Aα,Bβ and γ chains are expressed, assembled and secreted by hepatocytes as a hexamer (Aα,Bβ, γ)2 [11]. Fibrinogen chains are cotranslated into the lumen of the endoplasmic reticulum (ER) where folding and assembly is driven by the primary sequence with assistance of chaperones such as Bip and glycosylation enzymes [2]. Glycosylation begins in the ER and is finalized in the Golgi apparatus, where N-glycosylation of Bβ and γ is completed [2]. Before forming the full (Aα,Bβ, γ)2 molecule, each subunit assembles via heterodimer precursors, Aα/γ,Bβ/γ, to form half molecules where the Aα,Bβ and γ chains form triple helical coiled-coils, which are held together by disulphide bonds [2,12] (Figure1, panel A). Approximately 77% of synthesized fibrinogen is folded and secreted into the extracellular domain [2]. Misfolded or misassembled and surplus protein are retained in the ER and eventually undergo degradation by quality control mechanisms (lysosome and proteasome) [2]. Structural studies have shown that fibrinogen (Aα,Bβ, γ)2 assembles such that the Aα,Bβ, γ subunits are antiparallel to each other with the N termini of the subunits interacting with each other via disulphide bonds that hold the two trimeric subunits together to form the hexamer [13–17] (Figure1). As a result of its structural arrangement (Aα,Bβ, γ)2, the module consists of five regions; one central E region, two D regions that flank the region E and two outer αC regions (Figure1, panel B). Region E is the unique center that contains the N-termini of the six polypeptide chains. The D region comprises of a triple helical coiled coil referred to as the coiled-coil connector and the β- and γ-nodules (Figure1, panel B). The coiled-coil connectors connect region E to the β- and γ-nodules of region D. The αC region consists solely of the C-terminus of the Aα chain and comprises of an αC connector and αC region. Part of the αC connector folds back into the coiled coil connector through an alpha helix. Int.Int. J. J. Mol. Mol. Sci. Sci. 20212021,, 2222,, x 6916 FOR PEER REVIEW 33 of of 20 20 FigureFigure 1. AssemblyAssembly and and structure structure of fibrinogen. fibrinogen. ( (PanelPanel A A)) Production Production of of fibrinogen fibrinogen in hepatocytes. Once Once synthesised, synthesised, fibrinogenfibrinogen chains chains A Aαα, B,Bβ βandand γ assembleγ assemble in ain stepwise a stepwise manner. manner. The A Theα/γ and Aα/ Bγβand/γ heterodimers Bβ/γ heterodimers are formed are first, formed followed first, byfollowed the (Aα by/B theβ/γ) (A trimericα/Bβ/ subunit.γ) trimeric Once subunit. the trimeric Once thesubunits trimeric are subunits formed, arethey formed, dimerise they in dimerisean antiparallel in an antiparallelfashion to form fashion the (Aα/Bβ/γ)2 hexamer. (Panel B) shows a model of the fibrinogen structure based on the crystal structure of fibrinogen (PDB:3ghg) to form the (Aα/Bβ/γ)2 hexamer. (Panel B) shows a model of the fibrinogen structure based on the crystal structure of and NMR structure of the αC domain (PDB:2BAF). The assembly of the (Aα/Bβ/γ)2 hexamer gives rise to five regions, the E fibrinogen (PDB:3ghg) and NMR structure of the αC domain (PDB:2BAF). The assembly of the (Aα/Bβ/γ) hexamer gives region, two D regions and two αC regions. The E region is the central nodule that comprises of the N-termini 2of all the chains α (Ariseα shown to five in regions, blue, B theβ shown E region, in green two and D regions γ shown and in twored). TheC regions. D region The comprises E region of is a thetriple central coiled nodule coil connector that comprises and the β of- andthe N-terminiγ- nodules. ofThe all α theC domain chains composed (Aα shown of inthe blue, Aα chain Bβ shown and comprises in green of and theγ αshownConnector in red). and TheαC domain. D region comprises of a triple coiled coil connector and the β- and γ- nodules. The αC domain composed of the Aα chain and comprises of the αConnector and αC domain.2. The Biological Role of Fibrinogen (Conversion of Fibrinogen to Fibrin) Fibrinogen is a multifaceted protein with roles in tissue injury, inflammation, angio- genesis,2. The Biological cell migration Role and of Fibrinogen cell adhesion (Conversion [18–21]. This of Fibrinogen review will to focus Fibrin) on the role of fibrin(ogen)Fibrinogen in clot is aformation multifaceted and proteinlysis.
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